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sensors for living cells †
Guillaume Despras, Alsu I. Zamaleeva, Lucie Dardevet, Céline Tisseyre, Joao
Gamelas Magalhaes, Charlotte Garner, Michel de Waard, Sebastian
Amigorena, Anne Feltz, Jean-Maurice Mallet, et al.
To cite this version:
Guillaume Despras, Alsu I. Zamaleeva, Lucie Dardevet, Céline Tisseyre, Joao Gamelas Magalhaes, et
al.. H-Rubies, a new family of red emitting fluorescent pH sensors for living cells †. Chemical Science
, The Royal Society of Chemistry, 2015, 6, pp.5928-5937 �10.1039/c5sc01113b�. �hal-01197099�
H-Rubies, a new family of red emitting
fluorescent
pH sensors for living cells
†
Guillaume Despras,‡aAlsu I. Zamaleeva,‡bf
Lucie Dardevet,cdC´eline Tisseyre,cd Joao Gamelas Magalhaes,fCharlotte Garner,aMichel De Waard,cde
Sebastian Amigorena,f Anne Feltz,bJean-Maurice Malletaand Mayeul Collot§*a
Monitoring intracellular pH has drawn much attention due to its undeniably important function in cells. The widespread development offluorescent imaging techniques makes pH sensitive fluorescent dyes valuable tools, especially red-emitting dyes which help to avoid the overcrowded green end of the spectral band. Herein, we present H-Rubies, a family of pH sensors based on a phenol moiety and a X-rhodamine fluorophore that display a bright red fluorescence upon acidification with pKavalues spanning from 4 to
9. Slight structural modifications led to dramatic changes in their physicochemical properties and a relationship between their structures, their ability to form H-aggregates, and their apparent pKawas
established. While molecular form H-Rubies can be used to monitor mitochondrial acidification of glioma cells, their functionalised forms were linked via click chemistry to dextrans or microbeads containing a near infrared Cy5 (Alexa-647) in order to provide ratiometric systems that were used to measure respectively the phagosomal and endosomal pH in macrophages (RAW 264.7 cells) usingflow cytometry.
Introduction
Intracellular pH plays many different and important roles in cellular activity and is especially involved in ion transport,1 apoptosis,2,3 multidrug resistance4 and muscle contraction.5,6 Moreover, abnormal intracellular pH values are also associated with diseases such as Alzheimer's7 and cancer.8,9Monitoring variations of intracellular pH in living cells is therefore of the utmost importance and is an essential parameter for studying phagocytosis,10 endocytosis11 and consequently cellular
internalisation pathways. Fluorescence spectroscopy and uo-rescence imaging techniques have many advantages including high sensitivity, high spatial and temporal resolution and are not destructive to the cells. As such, molecularuorescent pH indicators have become indispensable tools for observing pH changes in cells. Although many uorescent indicators have already been developed (for an extensive review see: Han and Burgess),12several factors have to be taken into account in order to achieve efficient use within cells, including solubility in biological media, hydrophobicity, photostability and bright-ness. Our recent efforts to develop functionalisable red emitting uorescent sensors13–15were driven by the increasing use of cells transfected with green or yellowuorescent proteins (FPs), thus allowing multicolour imaging. Whilst recent progresses have been reported in the development of red emitting genetically encoded pH sensors,16 there is still a demand for efficient molecular red uorescent indicators. Among red-shied uo-rescent pH sensors, cyanines suffer from a weak photo-stability. Benzoxanthene dyes like SNARF, act as ratiometric probes but generally suffer from weak quantum yields. In contrast, red-emitting BODIPYs have recently attracted special attention due to their photostability and high brightness.17,18Having said this they tend to suffer from high lipophilicity making them ineffi-cient for cellular experiments as their apparent pKashis in
lipophilic environment.19
In his review, Han et al.12 pointed out that despite their advantageous spectral properties, pH probes based on rhoda-mine were not extensively developed.20Rhodamine-based pH aLaboratory of Biomolecules (LBM), UPMC Universit´e Paris 06, Ecole Normale
Sup´erieure (ENS), CNRS, UMR 7203, Paris F-75005, France. E-mail: mayeul.collot@ unistra.fr
bEcole Normale Sup´erieure, Institut de Biologie de l'ENS (IBENS), INSERM U1024,
CNRS UMR 8197, Paris F-75005, France
cInserm U836, LabEx Ion Channels, Science and Therapeutics, Grenoble Institute of
Neuroscience, chemin fortun´e ferrini, bˆatiment Edmond Safra, 38042 Grenoble Cedex 09, France
dUniversit´e Joseph Fourier, Grenoble, France eSmartox Biotechnology, Saint Martin d’H`eres, France fINSERM U932, Institute Curie, 75248, Paris, Cedex 05, France
† Electronic supplementary information (ESI) available: Details of the organic synthesis, characterisations (HPLC, NMR, mass spectra) as well as measurements of their photophysical properties can be found in the supplementary information. See DOI: 10.1039/c5sc01113b
‡ Contributed equally to this work.
§ Current address: Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Universit´e de Strasbourg, Facult´e de Pharmacie, 74, Route du Rhin, 67401, Illkirch, France.
Cite this: DOI: 10.1039/c5sc01113b
Received 27th March 2015 Accepted 13th July 2015 DOI: 10.1039/c5sc01113b www.rsc.org/chemicalscience
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probes mostly rely on the spirolactam ring opening principle, generally yielding pH sensors with high dynamic ranges but low pKavalues as well as high hydrophobicity due to their neutrally
charged OFF state.21–26
A key criteria for a pH sensor is its pKavalue, which should
be in the physiological range. While the cytosol and some other cellular compartments, such as the nucleus and endoplasmic reticulum maintain their pH at7.2, endosomes, lysosomes and secretory vesicles on the other hand are acidic environ-ments with the pH ranging from 4.5 to 6.7. Conversely, alkaline pH can be found in mitochondria (8),27 phagosomes of dendritic cells28,29and neutrophils.30As such, a collection of pH probes with a large range of pKavalues is required in order to
extend the scope of their application and thus a straightforward method of tuning their pKa values without tedious chemical
modications would be a necessity. Finally, the large majority of uorescent pH sensors do not offer an orthogonal and specic site of attachment required for functionalisation. Many common pH probes are too lipophilic and tend to compart-mentalise in hydrophobic domains of the cell, however, to date no alternative methods of functionalisation have been proposed. In this report we present the development of a new family of red-emitting pH probes based on X-rhodamines and a phenol moiety as the pH reporter (see abstract scheme). In addition to their pKavalues ranging from4 to 9, these ON–
OFF type pH sensors, based on the PET (photoinduced electron transfer) quenching phenomena, exhibit high brightness in their acidic phenol form and can display very high turn ON of theiruorescence thanks to the formation of dark soluble H-aggregates in their OFF state. Among this new family, a number of H-Rubies were designed to bear a linker allowing the func-tionalisation of dextran and microbeads in order to measure the pH in living cells.
Results and discussion
Synthesis of phenolic X-rhodamines
ON–OFF pH probes generally require a pH-sensitive moiety whereby protonation/deprotonation modies the quantum yield of a linkeduorophore either through PET (Photoinduced Electron Transfer) quenching phenomena or ICT (Internal Charge Transfer). Among these pH-sensitive moieties, phenol is the most popular as illustrated by the wide use ofuorescein and its derivatives: FITC, BCECF, etc. Moreover, the pKa of
phenols can be easily lowered and tuned to a biologically rele-vant range by introducing proper electron-withdrawing groups on the phenyl ring. Boens' group successfully converted an ortho-chlorophenol to obtain a pH-sensitive BODIPY with a pKa
of 7.6.31Only few examples of phenolic rhodamines are given in the literature32 and none of those were exploited as pH probes, leading us to investigate the potential efficiency of these entities as uorescent pH sensors. The red-emitting uorophore X-rhodamine was chosen for its highly desirable spectral properties including a high molar absorption, high quantum yield and good photo-stability as well as its reduced hydrophobicity compared to BODIPY. Thus, an initial set of 9 X-rhodamines was synthesised from hydroxybenzaldehydes
and hydroxynaphthaldehydes (for synthesis see ESI†). The use of phenyl-imidazol and (N-alkylated)-phenyl-pipera-zine33,34 as alternative pH-sensitive indicators was also explored giving rise to X-rhodamines: Imidazole, Pip-H and Pip-Alkyne (Fig. 1).
The spectral properties and pKavalues obtained for thisrst
set of synthesiseduorophores are given in Table 1. Although X-rhodamine Imidazole did not behave as a uorescent pH sensor (no signicant variation of uorescence intensity upon protonation), phenyl-piperazine and phenol-based X-rhoda-mines displayed clear pH sensitivity. Despite its physiologically relevant pKa value and the capacity to be functionalised,
Pip-Alkyne was not developed further as its dynamic range (3-fold) was found to be much lower than those obtained with phenol-based X-rhodamines.
Structure/properties relationship of H-Rubies
Absorption spectra of H-Rubies at different pH values provided us with information regarding their aggregation states (Fig. 2) since rhodamines can form different patterns of aggregations including H- and J-aggregates.35,36The observed broadening of absorption spectra for HR-pOH clearly indicated the formation of non-dened aggregates at high pH values. This could be explained by the equilibrium between the zwitterionic pheno-late form and the neutral, hydrophobic and planar quinone form (Fig. 2C). With regard to its isomers mOH and HR-oOH, the neutral quinone forms cannot be formed due to the impossible delocalization of the electrons in the HR-mOH form and a twisted structure imposed by hindered sterical effect in HR-oOH; therefore, their absorption spectra only display slight changes upon deprotonation with no sign of aggregation (Fig. 2A and B). Interestingly, absorption spectra of HR-Br at pH values above its pKa exhibited a second band indicating
formation of soluble H-aggregates (Fig. 2D). Basic forms of HR-Cl and HR-Br both form H-aggregates, the stabilisation of which could be attributed to intermolecular halogen bonding37,38 between the electron-rich oxygen of the phenolate and the halogen atom forming typical head to head H-aggregate dimers. It is also noted that H-Rubies that forms H-aggregates in their
Fig. 1 Structures of the synthesised X-rhodamines and thefirst set of H-Rubies.
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basic forms have signicantly enhanced acidity. For example, HR-Br is 1000-times more acidic than HR-pOH. This difference in acidity cannot be exclusively attributed to the electronic withdrawing effect of the bromine atom. These results tend to suggest that aggregation stabilises the basic forms of H-Rubies and hence, enhances their acidity. In this line of thought, since aggregation is concentration dependent, absor-bance spectra and titration curves of HR-Br were measured at different concentrations of dye ranging from 500 nM to 5 mM (see Fig. S3 and S4†). The results showed that aggregation of basic form occurred at concentration above 500 nM and impacted the pKawhich varied from 6.5 at 5mM to 7.3 at 500
nM. Moreover at a concentration of 500 nM where no aggre-gation of the basic form occurs theuorescence enhancement was diminished as the OFF state was only due to the PET quenching phenomenon. H-aggregates are virtually non-uo-rescent and thus, for the H-Rubies that are able to undergo this type of aggregation, this property can confer impressive levels ofuorescence enhancement (dynamic range) between their basic and acidic forms making them promising pH sensor for bio-imaging purposes. By controlling the H-aggregation ofuorophores one can achieve highly sensitive uorogenic systems with a pronounced turning ON of uo-rescence upon de-aggregation. This principle has been successfully used for bioimaging of ligand–receptor interac-tions39 and was also applied in monitoring intracellular pH withuorogenic polymers.40
Synthesis of functionalised H-Rubies
Despite therst phenol-based X-rhodamines yielding excellent results asuorescent pH probes, the importance of the ability to functionalise suchuorescent sensors must be emphasised and thus, the basic principle was pushed further in order to develop
Table 1 Spectral and physico-chemical properties of thefirst set of synthesised X-rhodamines (5 mM in MOPS 30 mM, 100 mM KCl)a
Compound
labsmax
(nm)
lemd
(nm)
Log3 (labsmax)
(M1cm1) F FON/FOFF Dynamic rangee Dynamic range @ pH 1 pKaf pKa HR-pOH ONb 576 587 4.78 0.55 9 34 30 8.97 OFFc 579f 598 4.53 0.06 0.05 HR-mOH ONb 578 602 4.70 0.30 3 132 75 9.33 OFFc 577 603 4,63 0.11 0.13 HR-oOH ONb 581 603 4.89 0.75 3 866 460 9.68 OFFc 579 603 4.69 0.27 0.17 HR-Me ONb 574 597 4.71 0.09 High 955 833 8.75
OFFc N/Ag 598 N/Ag N/Ah 0.16
HR-OMe ONb 577 598 4,72 0.22 8 302 N/Ai N/Ai
OFFc 576 600 4.74 0.03 HR-Cl ONb 579 601 4.68 0.47 High 753 512 6.17 OFFc 544 599 4.52 N/Ah 0.10 Hr-Br ONb 581 601 4.39 0.55 High 384 291 6.51 OFFc 544 600 4.40 N/Ah 0.10 p-Nph ONb 581 600 4.79 0.28 6 23 9.7 8.51 OFFc 579 602 4.34 0.05 0.17 o-Nph ONb 584 604 4.43 0.77 8 337 166 8.73 OFFc 576 604 4.46 0.10 0.17 Pip-H ONb 578 600 4.88 0.16 4 6 3.9 8.67 OFFc 579 601 4.84 0.04 0.07 Pip-Alkyne ONb 577 600 4.98 0.34 2 3 1.9 4.95 OFFc 579 602 4.45 0.15 0.09
Imidazole ONb 583 609 4.80 0.40 N/Aj N/Aj N/Aj N/Aj
OFFc 584 608 N/Aj N/Aj
a3 ¼ molar extinction coefficient, l ¼ wavelength, F ¼ quantum yield.bProtonated form: pH 4.cDeprotonated form: pH 10.dExcitation at 535 nm. e[(I
max Imin)/Imin] with I¼ uorescence intensity.fFluorescence enhancement between1 pH unit around the pKavalue.gBroad absorbance. hNon-uorescent.ipK
avalue was too high.jNo pH sensitivity.
Fig. 2 Absorption spectra of (A) HR-oOH, (B) HR-mOH, (C) HR-pOH and (D) HR-Br at 5mM in MOPS 30 mM, 100 mM KCl at different pH and their basic form (blue) and acidic form (red) structures.
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functionalisable H-Rubies. For this purpose, a linker was added at the ortho position of the phenol via an amide bond: a mild electron-withdrawing group that would ensure the conservation of a biologically relevant pKa. For this new set of
functionalis-able H-Rubies, 5-formylsalicylic acid 1 was converted into its activated ester 2 onto which were condensed primary and
secondary amines bearing one or two orthogonal functions. Once the desirable aldehydes were obtained, they were trans-formed into their corresponding X-rhodamines with a custom optimized methodology allowing us to obtain the H-Rubies with yields of up to 93% (see ESI†). This new set of sensors can be categorised as follows: alkynes and azides, allowing linkage via bio-orthogonal Huisgen cycloaddition“click chemistry”; acids that can be coupled with amines and bifunctional H-Rubies, bearing two different functional groups (Fig. 3). Among the bifunctional H-Rubies that were developed is an Fmoc-pro-tected Lysine H-Ruby (HR-LysF), which can be incorporated into a peptide sequence.
Structure/properties relationship of functionalisable H-Rubies
Once again, it was noticed that slight modications of the base structure led to dramatic changes in the photophysical prop-erties of H-Rubies such as epsilon, quantum yields and pKa
values which ranged from 4 to 8 (Table 2). The most notable differences were observed between secondary and tertiary amides whereby secondary amides appeared much more acidic than tertiary amides. To emphasise this phenomena, we syn-thesised HR-PN3 and its methylated tertiary amide equivalent
HR-MPN3and showed that the latter was 60-times less acidic.
Moreover, absorption spectra of these functionalisable H-Rubies clearly showed different behaviours of their basic forms in water. While deprotonated tertiary amide H-Rubies showed
Fig. 3 Synthesis and structures of the functionalisable H-Rubies: (a) NHS, DCC, THF; (b) amine, DIEA, DMSO; (c) 8-hydroxyjulolidine, PTSA, propionic acid then p-chloranil, DCM/MeOH (1 : 1); (d) 8-hydrox-yjulolidine, TfOH, DCM, thenp-chloranil.
Table 2 Spectral and physicochemical properties of the functionalisable H-Rubies (5mM in MOPS 30 mM, 100 mM KCl)
Compound
labsmax
(nm)
lemc
(nm)
Log3 (labsmax)
(M1cm1) F FON/FOFF Dynamic ranged Dynamic range @ pH 1 pKae pKa HR-A ONa 574 600 4.26 0.09 6 6 4.2 5.33 OFFb 534 598 4.58 0.02 0.13 HR-PA ONa 580 602 4.87 0.46 1150 31 22 6.89 OFFb 573 596 4.86 4 104 0.04 HR-PiA ONa 580 601 4.55 0.80 35 98 29 7.64 OFFb 574 600 4.32 0.02 0.10 HR-PiAC ONa 580 601 4.86 0.64 40 86 45 7.68 OFFb 575 602 4.69 0.02 0.09 HR-N3 ONa 580 600 4.43 0.25 28 88 74 3.96 OFFb 523 596 4.43 0.01 0.03 HR-PN3 ONa 578 603 4.72 0.41 21 21 19 6.20 OFFb 573 597 4.74 0.02 0.03 HR-MPN3 ONa 578 605 5.20 0.65 65 78 66 8.00 OFFb 573 603 5.20 0.01 0.03 HR-Ala ONa 577 601 4.81 0.15 8 9 8.3 7.85 OFFb 573 597 5.03 0.019 0.03
HR-bAla ONa 577 602 5.10 0.20 11 11 N/Af N/Af
OFFb 572 598 5.30 0.018
HR-LysF ONa 583 607 4.73 0.11 37 45 33 4.84
OFFb 580 600 4.66 0.003 0.10
HR-CysA ONa 577 602 4.69 0.09 N/Af N/Af N/Af N/Af
OFFb 572 597 5.08 0.014
HR-LysA ONa 577 602 4.75 0.25 13 13 11 7.51
OFFb 572 596 4.96 0.02 0.04
aProtonated form: pH 4.bDeprotonated form: pH 10.cExcitation at 535 nm.d[(I
max Imin)/Imin] with I¼ uorescence intensity.eFluorescence
enhancement between1 pH unit around the pKavalue.fComplex titration curve.
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slight hypsochromic shis of their maximum absorption wavelengths (1–5 nm), absorption spectra of secondary amide H-Rubies displayed a second absorption band (530 nm) indi-cating typical formation of soluble H-aggregates.
These differences can be explained by the formation of a H-bond between the phenolate and the proton of the secondary amide whereby the former is stabilised and hence, the pKais
lowered (Fig. 4). Moreover, the six membered ring formed via H-bonding leads to a constrained conformation which aids the formation of planar, head-to-head H-aggregates. As seen in our initial observations, the acidity of H-Rubies is enhanced by their ability to form H-aggregates in their OFF state (Fig. 4).
Imaging of acidic domains in living cells with molecular H-Rubies
The impressive physical and optical properties of H-Rubies lead us to investigate their ability to image acidic domains in living cells. Aer establishing their insensitivity towards biologically signicant metals (Zn2+, Mg2+, Ca2+, Fe3+, Mn2+) and their
nontoxicity (see ESI Fig. S5† for LDH cytotoxicity assay), HR-N3,
HR-Me, HR-Cl and HR-Br were involved in cellular experiments (Fig. 5A). These probes were specically chosen for their respective pKavalues: HR-Me served as a positive control since
its pKais more than two units above maximum physiological
pH, HR-N3was expected to be a negative control since it has a
pKa of 4, whereas HR-Cl and HR-Br were expected to sense
variations of pH within the cell (Fig. 5B). Firstly, Fischer's rat glioblastoma F98 cells (primary tumor) were incubated with the four dyes (1mM) for only 20 min and visualised by confocal laser scanning microscopy. The obtained images indicated that these H-Rubies are cell-permeant and stain the mitochondria; this was conrmed by colocalisation experiments with mitotracker-green (see Fig. S6†). In a second time, ow cytometry assays with these cells showed a clear correlation between the observed uorescence intensity of the cells and the measured pKaof the
probes (Fig. 5B and C plain lines). Finally, we checked the pH sensitivity of these H-Rubies upon cytosolic acidication. The intracellular pH was decreased to pH 6 and ow cytometry assays were performed (Fig. 5C dashed lines). The results showed that albeit HR-N3and HR-Cl displayed non-signicant
uorescence enhancement (blue and green lines), HR-Br (orange line) displayed an impressive in cellulo response to this acidication (DpH ¼ 1.4, uorescence enhancement: up to 13-fold). HR-Me also showed an interesting uorescence enhancement (5-fold) but behaved in an inconsistent manner when the intracellular pH was alkalinised (Fig. S7†). Despite HR-Br cannot be used to determine absolute pH values in cellulo because of the effect of its concentration on its pKa, its cell
permeability combined with its highuorescence enhancement in the physiological pH range make it an attractive tool for tracking mitochondrial acidication which is a important eld in cell biology as it is involved in mitophagy that is associated to various pathologies including neurodegenerative and cardio-vascular diseases.41,42
Measurements of intraorganellar pH using functionalisable H-Rubies
To demonstrate the different biological applications of H-Rubies we developed two systems to measure sub-organellar pH in living cells. Ourrst approach aimed at the calibration of phagosomal pH in living cells using ow cytometry. As pH reporter, HR-PiAC was chosen for its useful properties in cellular experiments such as its high brightness, its high dynamic range (80-fold) and its pKavalue of 7.68 (Fig. 6A and
B). 3mm latex beads containing amino groups were combined with the bifunctional linker azido-PEG4-NHS ester followed by
double functionalisation using click chemistry with HR-PiAC and a pH-insensitive near infrared emitting dye, Alexa Fluor 647, to allow ratiometric measurements (Fig. 6C). It was noticed that the covalent binding of two dyes on the surface of the beads
Fig. 4 Difference of acidity and H-aggregation capability between secondary amide H-Rubies (dashed lines) and tertiary amide H-Rubies (plain lines). Concentration of probes was 5mM in MOPS 30 mM, 100 mM KCl, excitation wavelength at 535 nm.
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inuences the physicochemical properties of HR-PiAC, namely its dynamic range and its pKa. This effect has already been
noticed in our previous work involving a molecularuorescent Ca2+sensor linked to a quantum dot43and can be attributed to the proximity of the dye to the hydrophobic environment of the polystyrene bead that enhances the basaluorescence of the sensor. Compared to the titration curve of the free dye, the uorescence ratio of the two dyes obtained by ow cytometry shows an almost linear dependence when the pH is varied from 5 to 9. As a result, although these beads have a decreased sensitivity in the pH of physiological domain, it provides a ratiometric system which displays a pH sensitivity over a wider range when compared to a free HR-PiAC (Fig. 6D).
The dyes-modied beads were then added to a suspension of RAW 264.7 macrophages and incubated for 30 min at 37C to complete the process of phagocytosis. Aer several washes the cells werexed and visualized by confocal microscopy to assess the efficiency of bead internalisation. Orthogonal view analysis of confocal sections shows partial internalisation of the beads by macrophages. As shown in Fig. 7, in addition to fully internalised beads, some beads are attached to the outer cell membrane. On average only 30% of beads were fully internal-ised by the cells. This could interfere with ow cytometric measurements of phagosomal pH because the laser-based
technique in this case will identify alluorescent objects, i.e. cells with internal and/or external beads. We, therefore, per-formed an additional modication of the beads by coating them with a biotinylated ovalbumin (OVA), via passive absorption. The coated beads,rstly, have a reduced non-specic binding to the cell surface and favour phagocytosis through the interaction between the mannosylated structure present on the surface of OVA44and the mannose receptors of macrophages.45Secondly, accessible biotin groups of OVA can be tagged by FITC-streptavidin.
For phagosomal pH measurement by ow cytometry, adherent RAW 264.7 macrophages were incubated for 30 min at 37C with the OVA-modied beads and let rest for 3 more hours for phagosomal maturation. Aerwards the cells were harvested and incubated with streptavidin-FITC that only binds to the non-internalised beads containing OVA-biotin. Two pop-ulations of phagocytic and non-phagocytic cells can be differ-entiated by theuorescence intensity of Alexa Fluor 647 of the beads. A cell population with beads attached to the cell surface
Fig. 5 (A) Structures and pKavalues of the H-Rubies used for cellular
experiments. (B) pH titration curves (Hill equationfit) of HR-N3(blue),
HR-Cl (green), HR-Br (red) and HR-Me (brown) at 5mM in MOPS (30 mM) KCl (100 mM), cytosolic and cellular acidic pH ranges are delimited with coloured areas. (C) Comparison of fluorescence intensity of F98 cells incubated for 20 min with 1mM of HR-N3(blue),
HR-Cl (green), HR-Br (red) and HR-Me (brown) at pH 7.4 (plain lines) and pH 6 (dashed lines) assessed byflow cytometry. Each curve is an average of 3 independent measurements. The control corresponds to the intrinsicfluorescence of cells (black line). Bottom: laser scanning confocal microscopy images of F98 cells incubated for 20 min with 1 mM of (D) HR-N3, (E) HR-Cl, (F) HR-Br, (G) HR-Me. The H-Rubies were
visualized in red, the nuclei were stained with Hoechst (blue) and the membrane with Alexa 647-conjugated concanavalin A (green). Pictures show the three merged channels. Scale bars are 10mm.
Fig. 6 Absorption (A) and emission spectra (B) of HR-PiAC (5mM, MOPS 30 mM, 100 mM KCl) at different pH (excitation wavelength ¼ 535 nm), inset is the titration curve fitted with the Hill equation providing a pKaof 7.68 0.09 and an enhancement of fluorescence of
80-fold. Scheme of bead-based pH sensor, containing pH-sensitive dye HR-PiAC and pH-insensitive Alexa Fluor 647 (C); titration curve of the pH beads measured byflow cytometry (D).
Fig. 7 Confocal microscopy images offixed RAW 264.7 cells after bead phagocytosis. DAPI nuclear staining, concanavalin-Alexa 488 membrane staining and pH beads are in blue, green and red respec-tively. (A) illustrates the orthogonal views of the cell that allow di ffer-entiation of the internalized beads (B) and the beads attached to the cell surface (C). Scale bars are 10mm.
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can be separated by the higheruorescence intensity of FITC at extracellular pH (Fig. 8A). Further detailed analysis of the phagocytic population shows the presence of subpopulations according to theuorescence intensities of Alexa Fluor 647 and H-Ruby: cells that have internalised one bead per cell exhibit a lower uorescence intensity (Fig. 8B). For precise analysis of phagosomal pH the cells were gated on one bead population and a ratiometric calibration curve of phagosomal pH was obtained changing the external pH over the 5 to 8 range using a citric acid/HEPES buffer and imposing the same pH intracel-lularly using nigericin, a K+/H+ionophore (Fig. 8C). Finally, to determine the phagosomal pH, the ratio of the mean uores-cence intensities of HRuby vs. Alexa Fluor 647 obtained from the intact cells was interpolated to the calibration curve yielding a phagolysosomal pH of 5.3 0.2. This result is in a good agreement with a previously published study where authors showed a phagosomal pH value of 5.22 0.03 in RAW cells using FITC-sRBC particles.46Addition of the weak base NH
4+/
NH3 caused fast alkalinisation, and demonstrated the
intra-cellular localisation of the beads (Fig. 8D).
Another application of these newly synthesized H-Rubies is the calibration of endosomal pH by ow cytometry and to achieve this we used a polysaccharide dextran that is known to target the endolysosomal pathway.4740 kDa dextran was con-verted to an alkyne containing dextran and was subsequently clicked to HR-PN3 (Fig. 9A). In comparison to its parent
molecular form HR-PN3, the uorescent pH-sensitive dextran
displayed a slightly shied apparent pKavalue (respectively 6.20
0.03 and 6.77 0.02; Fig. 9B) with a virtually non-uorescent OFF state (see ESI†). Functionalisation did not signicantly affect the uorescence enhancement between pH 10 and 4 (respectively 21- and 19-fold) making this water-soluble polymer a valuable tool for monitoring endosomal pH. To demonstrate this, adherent RAW 264.7 cells were incubated for 30 min with HR-PN3 dextran conjugate and let rest for 3 more hours. As
shown in Fig. 9C, vesicles of different shapes and sizes could be observed inside the cells due to the movement and segregation of the dextran molecules between the endocytic organelles.48
Fig. 8 Phagosomal pH measurement in RAW 264.7 cells byflow cytometry. (A) Density plot of the cells after bead internalisation showing three distinct populations: non-phagocytic cells, cells with internalised beads and cells with non-internalised beads on which a biotin– streptavidin– FITC interaction takes place; (B) the cells with only one internalised bead were used for analysis of phagosomal pH. (C) Typical calibration curve of H-Ruby/Alexa Fluor 647 ratio of the cells clamped at different pHs with the ionophore nigericin; (D) phagosomal pH (5.3 0.2, in red) in RAW 264.7 cells measured after 3 hours of phagosomes maturation NH4Cl addition shows alkalinisation of phagolysosomes (7.2 0.1, in blue). The
data represent the mean of 3 independent experiments SD.
Fig. 9 (A) Synthesis of HR-PN3dextran conjugatevia CuAAC Click
Chemistry. (B) Emission spectra of HR-PN3dextran conjugate (MOPS
30 mM, 100 mM KCl) at different pH (excitation wavelength ¼ 535 nm), inset is the titration curvefitted to the Hill equation yielding a pKaof
6.77 0.02 and an enhancement of fluorescence of 18-fold and 13 fold between1 pH unit around the pKa. (C–E) Confocal images of
RAW 264.7 cells after incubation with 1 mg mL1 HR-PN3 dextran
conjugate (C), co-stained by Lysotracker Green (D) and a merged image of two channels (E). HR-PN3dextran conjugate labels
lyso-somes (in red) that colocalises with Lysotracker, and endolyso-somes that are shown in insets and do not colocalise with Lysotracker. Scale bars are 10mm. (F) Typical calibration curve of H-Ruby/Alexa Fluor 647 ratio of the cells clamped at different pHs with the ionophore nigericin. (G) Endosomal pH (6.3 0.05, in red) in RAW 264.7 cells measured after 3 hours of incubation with HR-PN3 dextran. NH4Cl addition shows
alkalinisation of endosomal organelles (7.0 0.06, in blue). The data represent the mean of 3 independent experiments SD.
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Addition of Lysotracker Green led to its partial colocalisation with HR-PN3dextran conjugate (Fig. 9C–E) suggesting that
HR-PN3dextran accumulates both in the more acidic lysosomes and
in the mildly acidic vesicles of the endocytic pathway as well. This observation is consistent with theow cytometry experi-ments where, applying the same strategy as described above, the pH value of 6.3 0.04 was obtained (Fig. 9F–G).
Methods and materials
LDH-cytotoxicity assay
CHO-K1 cells (20 000 per well) were seeded in 96 multiple well plates 24 h before the experiment. Cells (40 000 per well) were incubated with theuorescent compounds in 100 mL DMEM for 2 hours at 37C. The incubation milieu was then replaced by 100mL fresh DMEM containing 10% of cell-counting solution (Dojindo Laboratories) and incubated for a further 2 h at 37C. The absorbance measured at 450 nm had a direct relationship to the number of viable cells. Experiments were conducted in triplicate and repeated twice.
Cell culture
Undifferentiated malignant glioma rat (F98) and Chinese Hamster Ovary (CHO) cell lines (from ATCC) were maintained at 37C in 5% CO2in DMEM/F-12 nutrient medium (Invitrogen,
Cergy Pontoise, France) supplemented with 2% (v/v) heat-inac-tivated fetal bovine serum (Invitrogen) and 100mg mL1 strep-tomycin and 100 units per mL penicillin (Invitrogen). RAW 264.7 macrophages cell line was maintained at 37C in 5% CO2
in DMEM nutrient medium (Gibco, Invitrogen) supplemented with 10% fetal calf serum (Eurobio, France).
Confocal microscopy
Cell cultures were incubated with the pH probes (in DMEM/F-12 nutrient medium only) for 20 min, and then washed twice with phosphate-buffered saline (PBS) alone. Aer 4 hours, the nucleus were stained with 60 mg mL1 Hoechst 34580 for 15 min, the cell cultures were then washed with PBS and the plasma membrane was stained with 30 mg mL1 Alexa 647-conjugated concanavalin A and the mitochondria stained with 50 nM mitotracker green for 5 min (stainings were purchased at Invitrogen, Cergy Pontoise, France). Cells were washed once more. Live cells were then immediately analysed by confocal laser scanning microscopy using a Zeiss LSM operating system. Alexa 647 (633 nm), Hoechst 34580 (405 nm), pH probes (561 nm) and mitotracker green (488 nm) were sequentially excited and emissionuorescence was collected.
To assess the efficiency of bead internalisation, the cells, aer completing the process of phagocytosis (as described in “Organellar pH measurement by ow cytometry”), were plated on coverslips (d¼ 12 mm) coated with 10 mg mL1bronectin and incubated at 37 C in 5% CO2: the cell membrane was
stained with 25mg mL1Alexa Fluor 488– conjugated conca-navalin (Invitrogen, USA) at 4 C for 5 min and 4% para-formaldehyde was added for 20 min to x the cells. The coverslips with the cells were then mounted on microscope
slides using a mounting medium DAPI-Fluoromount G (Southern Biotech, USA) and le at room temperature over-night. Imaging was performed on a spinning disk microscope. The pH-sensitive beads were excited with 561 nm laser line and detected with ET630/75 nmlter, DAPI was excited with 405 nm laser line and detected with ET460/50 nm band-passlter and Concanavalin A-Alexa Fluor 488 was excited with 488 nm laser line and detected with D505/40 nm band-pass lter. Images were collected using microscope soware Soware: MetaMorph 7.8.2.0. The subsequent analysis of the images was conducted on an open source image processing program ImageJ.
Fluorescence activated cell sorting analyses– effect of the pHi on theuorescence intensity of the H-Rubies
For the dose-response curve, F98 cells were incubated with various concentrations of the H-Rubies in DMEM/F-12 culture medium without serum at 37C for 2 h. The cells were then washed with PBS to remove excess extracellular H-Ruby and were then treated with 0.05% Trypsin–EDTA for 2 min at 37C
to detach cells from the surface, and centrifuged at 200 g in DMEM/F-12 culture medium before suspension in PBS. Flow cytometry analyses were performed with live cells using an Accuri C6 ow cytometer (BD Biosciences, Le Pont de Claix, France). For acquisition a 488 nm wavelength laser was used for H-Rubies excitation, theuorescence emissions were recorded in a 584 20 nm spectral detection channel. Data were obtained and analyzed using CFlow Sampler (BD Biosciences). Live cells were gated by forward/side scattering for a total of 10 000 events.
The pHi of F-98 cells was modied using the pseudo-null calibration method describe by Chow et al.49 F98 cells were incubated with 1 mM of H-Rubies in DMEM/F-12 culture medium without serum at 37C for 20 min. The cells were then washed with PBS to remove excess extracellular probe and were then treated with 0.05% Trypsin–EDTA for 2 min at 37C to
detach the cells from the surface, and centrifuged at 200 g in DMEM/F 12 culture medium before suspension in culture medium. Just before the analysis the pseudo null solution were added (ratio of culture medium: pseudo-null solution 1 : 1) and the data were acquired immediately (within 30 s). Pseudo-null solutions were made according to the method of Chow et al. Six times concentrated standard solution was made by adding 1 M ammonium hydroxide and 1 M acetic acid to the basal solution (culture medium). This provided a set of pseudo-null solution as shown in Table S1 (see ESI†). Flow cytometry analyses were performed with live cells using an Accuri C6ow cytometer (BD Biosciences, Le Pont de Claix, France). Data were obtained and analyzed using CFlow Sampler (BD Biosciences). Live cells were gated by forward/side scattering for a total of 10 000 events.
Functionalisation of the beads by H-Ruby
Polybead® Amino Microspheres 3.00 mm (Polysciences) were used. First the beads were washed with dH2O. In order to
convert the NH2groups on the beads surface to N3groups for
further click chemistry reactions, 100 nmol of the bifunctional linker N3–PEG4–NHS was added to 100 mL of the beads
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containing 20 nmol of NH2 groups and incubated at room
temperature for 2 h. The alkyne-modied H-Ruby HR-PiAC and alkyne-containing Alexa Fluor 647 (Molecular Probes®) were then mixed with the bead solution in a ratio 1 nmol of NH2
group: 10 nmol of H-Ruby: 2.5 nmol of Alexa Fluor 647. 5mL of 25 mM CuSO4$5H2O and 5mL of 25 mM sodium ascorbate were
added to initiate a click-reaction. The mixture was incubated overnight at room temperature. The dyes modied beads were successively washed with 0.1 M EDTA, 10% ethanol and PBS to remove non-reacted dyes.
Organellar pH measurement byow cytometry
Measurement of phagosomal or endosomal pH was performed using macrophages cell line RAW 264. The cells were plated on a Petri dish and at 80% conuence pH beads in a 4 : 1 ratio or 0.7 mg mL140 kDa HR-PN3dextran conjugate and 0.3 mg mL140
kDa Alexa Fluor 647 dextran conjugate were added and incu-bated 30 min at 37C to complete internalisation. Aerwards the non-internalised beads/dextran were washed out, fresh medium was added and the cells were let rest at 37C, 5% CO2
for 3 more hours. For FACS analysis the cells were harvested and resuspended in CO2-independent medium (GIBCO, Invitrogen).
For NH4+/NH3tests, 20 mM NH4Cl was added to the
extracel-lular medium and let to incubate for 3 min.
The calibration of phagosomal and endosomal pH was per-formed using the K+/H+ionophore nigericin that, together with a high concentration of potassium in the buffer, equalises the intracellular and extracellular pH.50 The cells containing internalised pH beads or dextran were resuspended in buffers containing 143 mM KCl, 1.17 mM MgCl2, 1.3 mM CaCl2, 5 mM
glucose and 10mM nigericin with dened pH buffers ranging from pH 5.0 to pH 8.0 with 0.5 increments. 20 mM citric acid was used for a buffer range of pH 5.0–6.5 and 20 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES) – for buffers in the pH range 7.0–8.0. 0.05 mM DAPI was added to exclude the dead cells. Aer incubation for 5 min at RT the samples were analyzed by aow cytometer (BD LSR Fortessa, BD Biosciences). For acquisition, a 532 nm wavelength laser was used for H-Rubies excitation, and its emissionuorescence was recorded in a 610 20 nm spectral detection channel. For Alexa Fluor 647 a 640 nm wavelength laser was used for excitation and a 670 30 nm spectral detection channel to record its emission uorescence. Each time 10 000 events were acquired. For analysis, a population of live cells was gated by forward/side scatter, and within this population only the cells which have phagocyted one pH bead were selected for precise pH determination.
Conclusion
We have developed a new set of efficient red-emitting uores-cent pH sensors based on PET quenching phenomena. H-Rubies exhibit specic pKa values and a structure/properties
relationship based on their ability to form non-emissive dark aggregates was established. Four non-functionalisable H-Rubies were chosen for cellular experiments and were shown to
cross the plasma membrane to compartmentalise in mito-chondria. Among them, HR-Br displays a stronguorescence enhancement upon acidication of mitochondria in live cells. Functionalisable H-Rubies were linked to a dextran polymer or to microbeads that were also decorated with a pH-insensitive dye leading to ratiometric pH probe systems. These systems were successfully used to measure phagosomal and endocytic pHs in live cells. The authors believe that this new family of uorescent indicators, with their large choice of pKavalues and
spectral properties specically adapted to cellular imaging, represents a valuable toolkit for imaging pH variations in living cells. Moreover, their diverse functionalisable side arms allows them to be attached to particles, polymers and biomolecules such as peptides or antibodies for accurate pH readout of subcellular micro-domains.
Acknowledgements
G. D. and A. Z. benetted of post-doctoral fellowships from the Pierre Gilles de Gennes foundation. C.T. was supported by the R´egion Rhˆone Alpes by an emergence fellowship. We would like to thank Jean-Marie Carpier (Institute Curie, U932) for his help with imaging, Dr. Sandrine Sagan and Dr Fabienne Burlina (Laboratoire des biomolecules, UMR CNRS 7203) for the toxicity assays. We also acknowledge PICT-IBiSA (Institute Curie), member of the France-BioImaging national research infra-structure. This work was supported by the Fondation Pierre-Gilles de Gennes (to S. A., A. F. and J. M. M.), the European Research Council (2013-AdG No. 340046), la Ligue Nationale contre le Cancer (EL 2014.LNCC/SA) and the French Agence National de la Recherche (ANR P3N, nanoFRET2 grant to A. F., M. D. W. and J. M. M., and ANR-11-LABX-0015 to M. D. W.). This work is the subject of a European patent EP 13 199 575.5– 1451 “uorescent red emitting functionalizable pH probes”.
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